1. Field of the Invention
The present invention relates to the field of nanoparticles, particularly carbon nanotubes and other nanoparticles and also to the field of cellular uptake of exogenous drugs or agents such as polypeptides, polynucleotide or small molecules with poor cellular uptake, and also to the fields of primary cell and T cell delivery.
2. Related Art
The interaction between nanostructured materials and living systems is of fundamental and practical interest and will determine the biocompatibility, potential utilities and applications of novel nanomaterials in biological settings. The pursuit of new types of molecular transporters is an active area of research, due to the high impermeability of cell membranes and other biological barriers to foreign substances and the need for intercellular delivery of molecules via cell-penetrating transporter for drug, gene or protein therapeutics (Henry, C. M. Chem. Eng. News 2004, 82, 37; Smith, D. A.; vandeWaterbeemd, H. Curr. Opion. Chem. Biol. 1999, 3, 373; Bendas, G. Biodrugs 2001, 15, 215). Recently, we and others (Kam, N. W. S.; Jessop, T. C.; Wender, P. A.; Dai, H. J. J. Am. Chem. Soc. 2004, 126, 6850; Pantarotto, D.; Briand, J.; Prato, M.; Bianco, A. Chem. Comm. 2004, 1, 16; Lu, Q.; Moore, J. M.; Huang, G.; Mount, A. S.; Rao, A. M.; Larcom, L. L.; Ke, P. C. Nano Lett. 2004, 4, 2473; Cherukuri, P.; Bachilo, S. M.; Litovsky, S. H.; Weisman, R. B. J. Am. Chem. Soc. 2004, 126, 15638; Bianco, A.; Hoebeke, J.; Godefroy, S.; Chaloin, O.; Pantarotto, D.; Briand, J.-P.; Muller, S.; Prato, M.; Partidos, C. D.; 16-Dec.-2004, W. R. D. J. Am. Chem. Soc. 2005, 127, 58) have uncovered the ability of single-walled carbon nanotubes (SWNTs) to penetrate mammalian cells and further transport various cargos inside cells including small peptides (Pantarotto), the protein streptavidin (Kam) and nucleic acids (Lu, Bianco). In our work of nanotube internalization and streptavidin transporting using nanotube carriers (Kam) and the work of Cherukiri et al., on nanotube uptake, the internalization mechanism was attributed to endocytosis. In the work of Pantarotto et al., Bianco et al., and Lu et al., nanotube uptake was suggested to be via insertion and diffusion through the lipid bilayer of cell membrane. While the uptake mechanism is unclear, it has been consistently reported that well-processed water-soluble nanotubes exhibit no apparent acute cytotoxicity to all living cell lines investigated thus far.
Covalent and non-covalent sidewall functionalization of single-walled carbon nanotubes (SWNT) has been actively pursued in recent years (Acc. Chem. Res. 2002, 35, Special issue on carbon nanotubes), aimed at several important goals. The first is to impart solubility to nanotubes in various solvents needed for dispersion, manipulation, sorting and separation. The second is to impart chemical functionality to nanotubes by attaching organic, inorganic or biological species to facilitate the interfacing of nanotubes with other materials for useful composites or bioconjugates. Functionalization of SWNTs with highly hydrophilic groups has been sought after in order to render nanotubes soluble in aqueous solutions. This would allow interfacing nanotubes with biological systems, potentially leading to an understanding of the biocompatibility (Mattson, M. P.; Haddon, R. C.; Rao, A. M. J. Mol. Neurosci. 2000, 14, 175-182) of nanotubes and the development of interesting biological applications including biosensors (Chen, R.; Zhang, Y.; Wang, D.; Dai, H. J. Am. Chem. Soc. 2001, 123, 3838-3839; Chen, R. J.; Bangsaruntip, S.; Drouvalakis, K. A.; Kam, N. W. S.; Shim, M.; Li, Y. M.; Kim, W.; Utz, P. J.; Dai, H. J., “Noncovalent functionalization of carbon nanotubes for highly specific biosensors,” Proc. Nat. Acad. Sci. USA. 2003, 100, 4984-4989). Various methods have been reported on modifying the sidewalls of the SWNTs to achieve solubility in water, including covalent functionalization by acid treatment (Chen, J.; Hammon, M. A.; Hu, H.; Chen, Y. S.; Rao, A. M.; Eklund, P. C.; Haddon, R. C. Science 1998, 282, 95-98), and physical adsorption of polymers, surfactants and nucleic acids (Chen, R. J.; Bangsaruntip, S.; Drouvalakis, K. A.; Kam, N. W. S.; Shim, M.; Li, Y. M.; Kim, W.; Utz, P. J.; Dai, H. J. Proc. Nat. Acad. Sci. USA. 2003, 100, 4984-4989; O'Connell, M. J.; Boul, P.; Ericson, L. M.; Huffman, C.; Wang, Y.; Haroz, E.; Kuper, C.; Tour, J.; Ausman, K.; Smalley, R. E. Chem. Phys. Lett. 2001, 342, 265-271; Moore, V. C.; Strano, M. S.; Haroz, E. H.; Hauge, R. H.; Smalley, R. E. Nano Lett. 2003, 3, 1379-1382).
It has now been surprisingly found that non-covalent functionalization of SWNTs can be accomplished by binding proteins to the nanotubes by various mechanisms, including strong adsorption of phospholipids grafted with polyethylene glycol (PEG) chains, which renders the nanotubes highly water-soluble. Previously, PEG-phospholipids (PEG-PL) have been investigated in the formation of micelles and liposomes for drug delivery (Adlakha-Hutcheon, G.; Bally, M. B.; Shew, C. R.; Madden, T. D. Nature Biotech. 1999, 17, 775-779; Meyer, O.; Kirpotin, D.; Hong, K.; Sternberg, B.; Park, J. W.; Woodle, M. C.; Papahadjopoulos, D. J. Biol. Chem. 1998, 273, 15621-15627; Papahadjopoulos, D.; Allen, T. M.; Gabizon, A.; Mayhew, E.; Matthay, K.; Huang, S. K.; Lee, K. D.; Woodle, M. C.; Lasic, D. D.; Redemann, C.; Martin, F. J. Proc. Nat. Acad. Sci. USA. 1991, 88, 11460-11464). In a recent work, Norris and coworkers have shown that PEG-PLs are excellent surfactants for solubilizing CdSe nanocrystals in aqueous phases Dubertret, B.; Skourides, P.; Norris, D. J.; Noireaux, V.; Brivanlou, A. H.; Libchaber, A. Science 2002, 298, 1759-1762).
Bianco EP 1 605 265, (published Dec. 14, 2005), “Non-covalent complexes comprising carbon nanotubes,” relates to the use of a carbon nanotube comprising positive and/or negative charges, said charges being carried by at least one charge-carrying group, said charge-carrying group being covalently bound to the surface of said carbon nanotube. Bianco further describes several non-covalent complexes between carbon nanotubes and various molecules, such as DNA or proteins, which have been described in the prior art. In most instances, the molecules and the carbon nanotubes are bound together through hydrophobic and/or π-stacking interactions.
Zheng, et al., (2003) Nature Mater. 2: 338-342 describe the solubilization of carbon nanotubes by single stranded DNA molecules, wherein the DNA molecule wraps helically around the carbon nanotube through π-stacking interactions to form a soluble complex. See also, Zheng, et al., “Structure-Based Carbon Nanotube Sorting by Sequence-Dependent DNA Assembly,” Science 29:1545-1548 (November 2003).
Dai, et al., WO 02/095099 (published Nov. 28, 2002 and related to PNAS 100:4984 cited above, as well as US PGPUB 2005/0100960) relates to complexes formed from the irreversible adsorption of molecules to the sidewalls of carbon nanotubes through π-stacking, van der Waals and hydrophobic interactions. As shown in FIG. 1 of the US PGPUB 2005/0100960, plurality of noncovalently-bonded molecules, having a highly aromatic group such as a pyrenyl group, are configured and arranged for bonding to additional molecules, such as biomolecules such as antibodies, antigens and DNA. These complexes are intended for in vitro use, e.g., as biosensors, where the attached molecules do not dissociate from the nanotubes.
Chen et al., PNAS 100:4989 (2003) shows the binding of various proteins (Steptavidin, avidin, BSA, staphylococcal protein A and α-glucosidase) to as-grown nanotubes, and nanotubes treated with surfactants such as Tween, Pluronic P103 and Triton-X. It was reported that a monolayer of Tween 20 anchored on a nanotube would repel non-specific binding of proteins in solution. Ten different polypropylene oxide molecules were investigated for their ability to adsorb onto nanotube walls.
One drawback associated with such complexes is that the dissociation of the complex is either difficult and/or has not been soundly assessed.
Another drawback associated to those complexes is that once dissociated from the complex, the carbon nanotube by itself is not soluble in aqueous systems and tends to form hydrophobic aggregates, which precipitate. Besides, non-functionalized carbon nanotubes have been shown to be toxic in several instances (Warheit et al., (2004) Toxicological Sciences 77:117-125; Lam et al., (2004) Toxicological Sciences 77: 126-134; Shvedova et al., (2003) Journal of Toxicology and Environmental Health, Part A 66:1909-1926).
Bianco et al., “Functionalized Carbon Nanotubes, A Process For Preparing the Same and Use in Medicinal Chemistry,” WO 2004/089818 (published 21 Oct. 2004) discloses functionalized carbon nanotubes of the general formula [Cn]—Xm where Cn are surface carbons of a carbon nanotube, X is a functional group and n and m are integers such that there are from about 2.10−11 to about 2.10−9 moles of X per cm2 of nanotube surface. The surface atoms Cn are reacted to form, as X, a pyrrolidine ring, bound to the nanotube by two different C—C bonds.
Hannah, US PGPUB 2004/0110128, published Jun. 10, 2004, entitled “Carbon Nanotube Molecular Labels,” discloses that carbon nanotubes may be derivatized with reactive groups to facilitate attachment to analytes or probes. Nanotubes may be derivatized to contain carboxylic acid groups (U.S. Pat. No. 6,187,823). Carboxylate derivatized nanotubes may be attached to nucleic acid probes or other analytes by standard chemistries, for example by carbodiimide mediated formation of an amide linkage with a primary or secondary amine group located on a probe or analyte. The methods of derivatization and cross-linking are not limiting and any reactive group or cross-linking methods known in the art may be used.
US PGPUB 20040038251 to Smalley, et al., published Feb. 26, 2004, entitled “Single-wall carbon nanotubes of precisely defined type and use thereof,” discloses coating nanotubes with a possibly toxic surfactant to prevent interaction with other nanotubes, and that the surfactant may be BRIJ® surfactants (BRIJ is a registered trademark of ICI Americas, Inc.; examples of BRIJ surfactants are polyethylene glycol dodecyl ether, polyethylene glycol lauryl ether, polyethylene glycol hexadecyl ether, polyethylene glycol stearyl ether, and polyethylene glycol oleyl ether), PLURONIC® Surfactants (PLURONIC is a registered trademark of BASF Corporation); and other surfactants.
Dwyer, et al., “DNA functionalized single-walled carbon nanotubes,” Nanotechnology 13:601-604 (2002) discloses linking DNA to nanotubes through amino-terminated DNA strands. A lambda DNA cluster is shown attached to a defect site and ends of an SWNT bundle.
Felekis, et al., “Single-walled carbon nanotube-based hybrid materials for managing charge transfer processes,” Rev. Adv. Mater. Sci. 10:272-276 (205) discloses formation of nanohybrids consisting of SWNT units and electron donor moieties such as porpyrinic and ferrocenyl units.
Menna et al., in a conference paper dated Oct. 1, 2003, “Shortened single-walled nanotubes functionalized with poly(ethylene glycol): preparation and properties,” disclose the grafting of PEG onto SWNTs after acid oxidative cutting, treatment with SOCl2 to yield SWNT-COCL, and amidation with PEG-monoamine.